The present invention relates generally to filtration devices and more particularly to microfabricated filters utilizing bulk substrate and thin film structures. The present invention further relates to biological containment wells and capsules for immunological isolation of cell transplants that are constructed with such filters.
Filtration devices are an indispensable necessity in the health care industry. Within the health care industry, accurate filtration devices are required, for example, in the fields of pharmaceutical technology, biotechnology, bioseparation, which includes plasma fractionation, and diagnostics. For many applications within these areas, required filtration device features include: precise control of pore sizes and distributions, absolute pore sizes as small as the nanometer (nm) range, mechanical strength, chemical inertness, high throughput, and high durability.
Filtration devices may, in particular, be utilized in containment wells and containment capsules constructed with such wells. Containment capsules are used for the immunological isolation of cell transplants. Containment wells may be used individually to evaluate the biological compatibility of different materials and to simulate the environment of containment capsules.
In a cell transplant, cells from a donor are transplanted into a host. The donor cells generate biologically-active molecules that provide the needed metabolic function in the host. As an example of a cell transplant, the islets of Langerhans, which produce insulin in mammals, have been transplanted between different species. However, unprotected islets function only for a short time before the immune system of the host kills the donor cells.
Encapsulation of islets in order to protect them from immune system macromolecules has been shown to prolong the survival of donor cells. For instance, by using various means of encapsulation, insulin production from pig islets has been maintained for over one hundred days in dogs. Encapsulation methods to date have used semipermeable amorphous organic polymeric membranes. Significant problems have been encountered, however, limiting the useful life of these capsules to not much more than one hundred days.
One problem with organic membrane capsules is inadequate mechanical strength. If the thickness of an organic membrane capsule wall is increased to provide the required mechanical strength, the biologically-active molecules cannot diffuse through the capsule wall quickly enough to provide the appropriate physiological response when needed.
Another problem with these capsules is insufficient control of pore size and pore distribution. If the size and distribution of pores cannot be controlled, such as with amorphous polymeric membranes, there is a high probability of oversized or overlapping pores which could provide an opening large enough for immunological macromolecules to enter the capsule.
Yet another problem with organic membrane capsules is their lack of biological compatibility and chemical inertness. The pores of the capsule membrane are susceptible to clogging by immunocytes, thus triggering an immune response in the host. The capsule membrane is also prone to adsorption of molecules such as proteins, causing the pores of the membrane to become clogged and thus restricting the passage of biologically-active molecules through the capsule wall. Furthermore, the capsule membrane is water soluble, thus limiting the capsule's useful life.
Precise control of filter pore sizes in the 5 to 20 nm range would allow biologically-important molecules to be mechanically separated on the basis of size. For instance, such pore sizes may be used to achieve the heretofore elusive goal of viral elimination from biological fluids. In the present state of the art, there is a very limited selection of filters having pore sizes much less than the resolution limit of photolithography, currently 0.35 micrometers (.mu.m). The filters known heretofore having pore sizes in this range include polycarbonate membrane filters, sintered filters, zeolites, and microfabricated micromachined filters.
Polycarbonate membrane filters (nucleopore filters) may be used where pore sizes between 50 and 350 nm are needed. These filters, however, cannot be used at high temperatures, in strong organic solvents, or where no extracted oligomers can be tolerated. The pores of polycarbonate membrane filters are also randomly located. As such, there is a compromise between having a high enough population of pores per unit area and having too many instances of partially overlapping pores. Partially overlapping pores provide pathways through the filter that allow some particles to get through that are larger in diameter than the rated cut-off size of the filter.
Filters that are available in other materials, such as metals or ceramics, are made by sintering together discrete particles. This technique yields a random structure with a relatively large dead volume and no exact cut-off size above which transport is impossible.
Materials such as zeolites, which have a crystal structure with large channels, can be used as molecular sieves in the limited range of from about 0.5 nm to 5 nm. Zeolites are not amenable, however, to fabrication as thin membranes and thus provide a relatively low throughput.
A microfabricated filter comprised of surface micromachined structures is described in U.S. patent application Ser. No. 08/207,457, filed on Mar. 7, 1994, now U.S. Pat. No. 5,651,900, and assigned to the assignee of the subject application. The filter yields relatively uniform pore sizes and distributions. The pore sizes can be as small as about 5 nm. The walls of the filtration channels of the filter are entirely composed of polycrystalline silicon.
A microfabricated filter with a combination of surface and bulk micromachined structures is described by Kittilsland in Sensors and Actuators, A21-A23 (1990) pp. 904-907. Unlike the previously described microfabricated filter, the filtration channels of this filter are partially composed of single crystalline silicon. Single crystalline silicon has an improved mechanical strength and chemical inertness over amorphous or polycrystalline silicon. As a result, the filter is relatively resistant to the adsorption of particles within a solution, such as protein molecules, that may clog its pores.
The pores of the filter described in Kittilsland are defined by diffusing regions of boron into the single crystalline silicon substrate through a silicon dioxide mask and then etching away the portions of the substrate that are not doped with boron. The length of the pores is determined by the extent of lateral diffusion of the boron through the substrate.
The filter of Kittilsland suffers from several disadvantages as a result of being fabricated by this process. First, the mechanical strength of the filter cannot be increased without increasing the length of the pores. This is because the thickness of the etched substrate and the length of the pores are both dependent on the diffusion of boron into the substrate and cannot be controlled independently of each other. Second, the pore length of the filter is not tightly controlled. This is because the rate of lateral diffusion of boron into the substrate, which affects the pore length, cannot be precisely regulated. Third, the fabrication process cannot be used to construct a filter with in-line pores, i.e., pores that have channels aligned in the same direction as the liquid flow. Fourth, the bulk micromachined structure of the filter has a non-uniform thickness, thus reducing the filter's mechanical strength. This results from the hemispherical shape of the regions of boron diffused into the substrate. Fifth, the density of pores in the filter is limited. This is again due to the use of regions of boron diffusion to define the pores.
An improved filter should combine mechanical strength with the ability to allow the free diffusion of small molecules such as oxygen, water, carbon dioxide, and glucose, while preventing the passage of larger molecules such as the immunoglobins and major histocompatibility (MHC) antigens. Furthermore, such a filter should allow the diffusion of intermediate sized molecular products, such as insulin, at a sufficient rate to enable a containment capsule utilizing such a filter to provide the needed metabolic function in the host. The filter should also be resistant to the adsorption of molecules. Finally, the filter should provide a high throughput or flow rate per unit area. A high throughput may be provided by constructing the filter out of a very thin membrane. Throughput may also be improved by utilizing in-line pores.
Containment capsules utilizing such filters would have a longer life than presently-available capsules, and eliminate the need for anti-rejection drugs by the simple strategy of physically isolating the transplanted cells so that no immunological reaction can take place. Cells from any source could then be implanted in any host. Tissue matching of donor to recipient would not be a concern.
The ideal filter material for such capsules would be biologically compatible and chemically inert with sufficient mechanical strength to form a very thin membrane having at least a region with uniformly sized and spaced holes that are just large enough to let the desired biologically-active molecular product through, while totally blocking the passage of all larger immunological molecules. Such a structure cannot be made from a polymer with an amorphous molecular structure, by sintering together particles, or by intermeshed ceramic needles.
Accordingly, it is an object of the present invention to provide a filter having a precisely controlled pore width, length and distribution.
It is another object of the present invention to provide a filter having a very short pore width and length, with the pore width in the nanometer range.
Yet another object of the present invention is to provide a filter having a relatively high throughput.
An additional object of the present invention is to provide a filter that is made of a biologically compatible, chemically inert material having a mechanical strength sufficient to form a very thin membrane.
A further object of the present invention is to provide methods for the construction of such a filter using relatively simple fabrication techniques.
Yet another object of the present invention is to provide a containment well or capsule constructed with such a filter to let a desired biologically-active molecular product through the well or capsule at a physiologically desirable rate, while blocking the passage of all larger immunological molecules, thus providing an immunological isolation of cell transplants contained therein.
It is another object of the present invention to provide methods for the construction of such a containment well or capsule using relatively simple fabrication techniques.
Still another object of the present invention is to provide methods for administering a biologically-active molecule to a host organism deficient in endogenous production of said biologically-active molecule using such a containment capsule.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the claims.